Abstract:

Abstract:Gene editing technology is a genetic operation technology that can modify the DNA sequence at the genomic level. The precision gene editing technology based on CRISPR/Cas9 system is a gene editing technology that is easy to operate and widely used. Unlike the traditional CRISPR/Cas9 system, the precision gene editing technology can perform site-directed mutation of genes without DNA template. This review summarizes the recent development of precision gene editing technology based on CRISPR/Cas9, and prospects the challenges and opportunities of this technology.

Abstract:Color is an important indicator for evaluating the ornamental traits of horticultural plants, and plant pigments is a key factor affecting the color phenotype of plants. Plant pigments and their metabolites play important roles in color formation of ornamental organs, regulation of plant growth and development, and response to adversity stress. It has therefore became a hot topic in the field of plant research. Virus-induced gene silencing (VIGS) is a vital genomics tool that specifically reduces host endogenous gene expression utilizing plant homology-dependent defense mechanisms. In addition, VIGS enables characterization of gene function by rapidly inducing the gene-silencing phenotypes in plants. It provides an efficient and feasible alternative for verifying gene function in plant species lacking genetic transformation systems. This paper reviews the current status of the application of VIGS technology in the biosynthesis, degradation and regulatory mechanisms of plant pigments. Moreover, this review discusses the potential and future prospects of VIGS technology in exploring the regulatory mechanisms of plant pigments, with the aim to further our understandings of the metabolic processes and regulatory mechanisms of different plant pigments as well as improving plant color traits.

Abstract:High salt content in soils severely hampers plant growth and crop yields. Many transcription factors in plants play important roles in responding to various stresses, but their molecular mechanisms remain unclear. WRKY transcription factors are one of the largest families of transcription factors in higher plants that are involved in and influence many aspects of plant growth and development. They play important roles in responding to salt stress. The regulation of gene expression by WRKY proteins is mainly achieved by binding to the DNA's specific cis-regulatory elements, the W-box elements (TTGACC). In recent years, there have been many studies revealing the roles and mechanisms of WRKY family members, from model plant Arabidopsis to agricultural crops. This paper reviews the latest research progress on WRKY transcription factors in response to salt stress and discusses the current challenges and future perspectives of WRKY transcription factor research.

Abstract:Excessive levels of cadmium (Cd) in soil exert serious negative impacts on soil ecosystems. Microorganisms are a common component of soil and show great potential for mitigating soil Cd. This review summarizes the application and remediation mechanisms of microorganisms, microbial-plants, and microbial-biochar in Cd-contaminated soil. Microorganisms such as Bacillus, Acinetobacter, Pseudomonas, and arbuscular mycorrhizal fungi (AMF) can change the biological validity of Cd through adsorption, mineralization, precipitation and dissolution. Different factors such as pH, temperature, biomass, concentration, and duration have significant effects on Cd bioavailability by microorganisms. Pseudomonas, Burkholderia, and Flavobacterium can promote the uptake of Cd²⁺ by hyperaccumulator through promotion and activation. Biochar, a soil amendment, possesses unique physicochemical properties and could act as a shelter for microorganisms in agriculture. The use of combined microbial-biochar can further stabilize Cd compared to using biochar alone.

Abstract:Porcine epidemic diarrhea (PED) is a highly contagious disease that causes high mortality in suckling piglets. Although several licensed inactivated and live attenuated vaccines were widely used, the infection rate remains high due to unsatisfactory protective efficacy. In this study, mRNA vaccine candidates against PED were prepared, and their immunogenicity was evaluated in mice and pregnant sows. The mRNA PED vaccine based on heterodimer of viral receptor binding region (RBD) showed good immunogenicity. It elicited robust humoral and cellular immune responses in mice, and the neutralizing antibody titer reached 1:300 after a single vaccination. Furthermore, it induced neutralizing antibody level similar to that of the inactivated vaccine in pregnant sows. This study developed a new design of PED vaccine based on the mRNA-RBD strategy and demonstrated the potential for clinical application.

Abstract:The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl I digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.

Abstract:Nearly a quarter of the world's population is infected with Mycobacterium tuberculosis and remains long-term asymptomatic infection. Rv2626c is a latent infection-related protein regulated by DosR of M. tuberculosis. In this study, the Rv2626c protein was prokaryotically expressed and purified, and its immunobiological characteristics were analyzed using RAW264.7 cells and mice as infection models. SDS-PAGE and Western blotting analysis showed that the Rv2626c-His fusion protein was mainly expressed in soluble form and specifically reacted with the rabbit anti-H37RV polyclonal serum. In addition, we found that the Rv2626c protein bound to the surface of RAW264.7 macrophages and up-regulated the production of NO. Moreover, the Rv2626c protein significantly induced the production of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6 and MCP-1, and induced strong Th1-tendency immune response. These results may help to reveal the pathogenic mechanism of M. tuberculosis and facilitate the development of new tuberculosis vaccine.

Abstract:Somatostatin (SST) is an inhibitory polypeptide hormone that plays an important role in a variety of biological processes. Somatostatin receptor 2 (SSTR2) is the most widely expressed somatostatin receptor. However, the specific cell types expressing Sstr2 in the tissues have not been investigated. In this study, we detected the expression pattern of SSTR2 protein in mouse at different development stages, including the embryonic 15.5 days and the postnatal 1, 7, 15 days as well as 3 and 6 months, by multicolour immunofluorescence analyses. We found that Sstr2 was expressed in some specific cells types of several tissues, including the neuronal cells and astrocytes in the brain, the mesenchymal cells, the hematopoietic cells, the early hematopoietic stem cells, and the B cells in the bone marrow, the macrophages, the type II alveolar epithelial cells, and the airway ciliated cells in the lung, the epithelial cells and the neuronal cells in the intestine, the hair follicle cells, the gastric epithelial cells, the hematopoietic stem cells and the nerve fibre in the spleen, and the tubular epithelial cells in the kidney. This study identified the specific cell types expressing Sstr2 in mouse at different developmental stages, providing new insights into the physiological function of SST and SSTR2 in several cell types.

Abstract:The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested:the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P<0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P<0.05 or P<0.01); the expression of ACE2 decreased significantly (P<0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P<0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P>0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P<0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P<0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P>0.05), but the IL-6 level and expression of mRNA increased significantly (P<0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P>0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.

Abstract:The aim of this study was to clone the chicken zp1 gene encoding zona pellucida 1 (Zp1) and investigate its tissues expression profile and its effect on osteoblast mineralization. The expression level of zp1 was quantified in various tissues of laying hens and in the tibia of the pre- and post-sexual maturity by RT-qPCR. Zp1 overexpressed vector was transfected into chicken calvarial osteoblasts which were induced differentiation for 8 days, and the extracellular mineral and the expression of mineralization-related genes were detected. The full-length chicken zp1 gene is 3 045 bp, encoding 958 amino acids residuals, and has two N-glycosylation sites. The highest expression level of the zp1 gene was found in the liver, followed by the tibia and yolk membrane, while no expression was detected in the heart and eggshell gland. Compared with the pre-sexual maturity hens, the concentration of estrogen (E2) in plasma, the content of glycosaminoglycan (GAG) and the expression level of the zp1 gene in the tibia with post-sexual maturity were higher. The extracellular matrix and the level of osteoblast mineralization-related genes showed a significantly upregulated expression in chicken calvarial osteoblasts with Zp1 overexpressed and addition of estrogen. The expression of the zp1 gene is tissue-specific and positively regulated osteoblast mineralization under the action of estrogen, laying the foundation for elucidating the functional properties of Zp1 in chicken bones during the egg production period.

Abstract:The aim of this study was to clone the goat RPL29 gene and analyze its effect on lipogenesis in intramuscular adipocytes. Using Jianzhou big-eared goats as the object, the goat RPL29 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR), the gene structure and expressed protein sequence were analyzed by bioinformatics, and the mRNA expression levels of RPL29 in various tissues and different differentiation stages of intramuscular adipocytes of goats were detected by quantitative real-time PCR (qRT-PCR). The RPL29 overexpression vector pEGFP-N1-RPL29 constructed by gene recombination was used to transfect into goat intramuscular preadipocytes and induce differentiation. Subsequently, the effect of overexpression of RPL29 on fat droplet accumulation was revealed morphologically by oil red O and Bodipy staining, and changes in the expression levels of genes related to lipid metabolism were detected by qRT-PCR. The results showed that the length of the goat RPL29 was 507 bp, including a coding sequence (CDS) region of 471 bp which encodes 156 amino acid residues. It is a positively charged and stable hydrophilic protein mainly distributed in the nucleus of cells. Tissue expression profiling showed that the expression level of this gene was much higher in subcutaneous adipose tissue and inter-abdominal adipose tissue of goats than in other tissues (P<0.05). The temporal expression profile showed that the gene was expressed at the highest level at 84 h of differentiation in goat intramuscular adipocytes, which was highly significantly higher than that in the undifferentiated period (P<0.01). Overexpression of RPL29 promoted lipid accumulation in intramuscular adipocytes, and the optical density values of oil red O staining were significantly increased (P<0.05). In addition, overexpression of RPL29 was followed by a highly significant increase in ATGL and ACC gene expression (P<0.01) and a significant increase in FASN gene expression (P<0.05). In conclusion, the goat RPL29 may promote intra-muscular adipocyte deposition in goats by up-regulating FASN, ACC and ATGL.

Abstract:The evaluation of the bioavailability of pollutants in soil is crucial to accurately assess the pollution risk, and whole-cell biosensor is one of the important tools for such evaluation. This study aimed to develop a novel whole-cell biosensor for the detection of methyl parathion in soil using. First, a whole-cell biosensor was constructed by the screened methyl parathion hydrolase mpd gene, the existing specific induction element pobR, and the pUC19 plasmid skeleton. Then, the detection method of methyl parathion in soil extracts was established using 96-well microtiter plate as carrier and five whole-cell biosensors as indicator. The method was applied in the detection of methyl parathion in tested and field soil extracts. Taking E. coli DH5α/pMP-AmilCP with the best detection performance as an example, this biosensor had a detection limit of 6.21−6.66 µg/L and a linear range of 10−10 000 µg/L for methyl parathion in four soil extracts. E. coli DH5α/pMP-RFP and E. coli DH5α/pMP-AmilCP methods have good detection performance for the analysis of methyl parathion in soil extract samples. This biosensor method can help to quickly assess the bioavailability of methyl parathion in soil, and thus help to understand the risk of soil pollution caused by organophosphorus pesticide methyl parathion.

Abstract:To investigate the bioelectrochemical enhanced anaerobic ammonia oxidation (anammox) nitrogen removal process, a bioelectrochemical system with coupled anammox cathode was constructed using a dual-chamber microbial electrolysis cell (MEC). Specifically, a dark incubation batch experiment was conducted at 30℃ with different influent total nitrogen concentrations under an applied voltage of 0.2 V, and the enhanced denitrification mechanism was investigated by combining various characterization methods such as cyclic voltammetry, electrochemical impedance spectroscopy and high-throughput sequencing methods. The results showed that the total nitrogen removal rates of 96.9%±0.3%, 97.3%±0.4% and 99.0%±0.3% were obtained when the initial total nitrogen concentration was 200, 300 and 400 mg/L, respectively. In addition, the cathode electrode biofilm showed good electrochemical activity. High-throughput sequencing results showed that the applied voltage enriched other denitrifying functional groups, including Denitratisoma, Limnobacter, and ammonia oxidizing bacteria SM1A02 and Anaerolineaceae, Nitrosomonas europaea and Nitrospira, besides the anammox bacteria. These electrochemically active microorganisms comprised of ammonium oxidizing exoelectrogens (AOE) and denitrifying electrotrophs (DNE). Together with anammox bacteria Candidatus Brocadia, they constituted the microbial community structure of denitrification system. Enhanced direct interspecies electron transfer between AOE and DNE was the fundamental reason for the further improvement of the total nitrogen removal rate of the system.

Abstract:c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni²⁺-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells.The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.

Abstract:Nitrate is the main form of inorganic nitrogen that crop absorbs, and nitrate transporter 2 (NRT2) is a high affinity transporter using nitrate as a specific substrate. When the available nitrate is limited, the high affinity transport systems are activated and play an important role in the process of nitrate absorption and transport. Most NRT2 cannot transport nitrates alone and require the assistance of a helper protein belonging to nitrate assimilation related family (NAR2) to complete the absorption or transport of nitrates. Crop nitrogen utilization efficiency is affected by environmental conditions, and there are differences between varieties, so it is of great significance to develop varieties with high nitrogen utilization efficiency. Sorghum bicolor has high stress tolerance and is more efficient in soil nitrogen uptake and utilization. The S. bicolor genome database was scanned to systematically analyze the gene structure, chromosomal localization, physicochemical properties, secondary structure and transmembrane domain, signal peptide and subcellular localization, promoter region cis-acting elements, phylogenetic evolution, single nucleotide polymorphism (SNP) recognition and annotation, and selection pressure of the gene family members. Through bioinformatics analysis, 5 NRT2 gene members (designated as SbNRT2-1a, SbNRT2-1b, SbNRT2-2, SbNRT2-3, and SbNRT2-4) and 2 NAR2 gene members (designated as SbNRT3-1 and SbNRT3-2) were identified, the number of which was less than that of foxtail millet. SbNRT2/3 were distributed on 3 chromosomes, and could be divided into four subfamilies. The genetic structure of the same subfamilies was highly similar. The average value of SbNRT2/3 hydrophilicity was positive, indicating that they were all hydrophobic proteins, whereas α-helix and random coil accounted for more than 70% of the total secondary structure. Subcellular localization occurred on plasma membrane, where SbNRT2 proteins did not contain signal peptides, but SbNRT3 proteins contained signal peptides. Further analysis revealed that the number of transmembrane domains of the SbNRT2s family members was greater than 10, while that of the SbNRT3s were 2. There was a close collinearity between NRT2/3s of S. bicolor and Zea mays. Protein domains analysis showed the presence of MFS_1 and NAR2 protein domains, which supported executing high affinity nitrate transport. Phylogenetic tree analysis showed that SbNRT2/3 were more closely related to those of Z. mays and Setaria italic. Analysis of gene promoter cis-acting elements indicated that the promoter region of SbNRT2/3 had several plant hormones and stress response elements, which might respond to growth and environmental cues. Gene expression heat map showed that SbNRT2-3 and SbNRT3-1 were induced by nitrate in the root and stem, respectively, and SbNRT2-4 and SbNRT2-3 were induced by low nitrogen in the root and stem. Non-synonymous SNP variants were found in SbNRT2-4 and SbNRT2-1a. Selection pressure analysis showed that the SbNRT2/3 were subject to purification and selection during evolution. The expression of SbNRT2/3 gene and the effect of aphid infection were consistent with the expression analysis results of genes in different tissues, and SbNRT2-1b and SbNRT3-1 were significantly expressed in the roots of aphid lines 5-27sug, and the expression levels of SbNRT2-3,SbNRT2-4 and SbNRT3-2 were significantly reduced in sorghum aphid infested leaves. Overall, genome-wide identification, expression and DNA variation analysis of NRT2/3 gene family of Sorghum bicolor provided a basis for elucidating the high efficiency of sorghum in nitrogen utilization.

Abstract:Galactinol synthase (GolS) genes play important roles in plant response to abiotic stress. In this research, the plant expression vector of soybean GmGolS2-2 gene was constructed and transformed into tobacco to study the drought tolerance of transgenic tobacco. A GmGolS2-2 gene with 975 bp coding sequence was cloned from soybean leaves by reverse transcription-polymerase chain reaction (RT-PCR). GmGolS2-2 was linked to the plant expression vector pRI101 by restriction enzyme sites Nde I and EcoR I, and transformed into tobacco by leaf disc method. Genomic DNA PCR and real-time PCR showed that three GmGolS2-2 transgenic tobacco plants were obtained. The growth status of GmGolS2-2 transgenic tobacco under drought stress was better than that of wild-type tobacco. After drought stress treatment, the electrolyte leakage and malondialdehyde content of transgenic tobacco were lower than those of wild-type tobacco, but the proline content and soluble sugar content were higher than those of wild-type tobacco. The results of real-time PCR showed that the heterologous expression of GmGolS2-2 increased the expression of stress-related genes NtERD10C and NtAQP1 in transgenic tobacco. The above results indicated that GmGolS2-2 improved drought resistance of transgenic tobacco.

Abstract:Sweet potato is an important food crop that can also be used as an industrial raw material. Sucrose is the main form of long-distance carbohydrate transport in plants, and sucrose transporter (SUT) regulates the transmembrane transport and distribution of sucrose during plant growth and metabolism. Moreover, SUT plays a key role in phloem mediated source-to-sink sucrose transport and physiological activities, supplying sucrose for the sink tissues. In this study, the full-length cDNA sequences of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were obtained by rapid amplification of cDNA ends (RACE) cloning according to the transcripts of the two SUT coding genes which were differentially expressed in sweet potato storage roots with different starch properties. Phylogenetic analysis was performed to clarify the classification of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆. The subcellular localization of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ was determined by transient expression in Nicotiana benthamiana. The function of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in sucrose and hexose absorption and transport was identified using yeast functional complementarity system. The expression pattern of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in sweet potato organs were analyzed by real-time fluorescence quantitative PCR (RT-qPCR). Arabidopsis plants heterologous expressing IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ genes were obtained using floral dip method. The differences in starch and sugar contents between transgenic and wild-type Arabidopsis were compared. The results showed IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ encoded SUT proteins with a length of 505 and 521 amino acids, respectively, and both proteins belonged to the SUT1 subfamily. IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were located in the cell membrane and were able to transport sucrose, glucose and fructose in the yeast system. In addition, IbSUT₆₂₇₈₈ was also able to transport mannose. The expression of IbSUT₆₂₇₈₈ was higher in leaves, lateral branches and main stems, and the expression of IbSUT₈₁₆₁₆ was higher in lateral branches, stems and storage roots. After IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ were heterologously expressed in Arabidopsis, the plants grew normally, but the biomass increased. The heterologous expression of IbSUT₆₂₇₈₈ increased the soluble sugar content, leaf size and 1 000-seed weight of Arabidopsis plants. Heterologous expression of IbSUT₈₁₆₁₆ increased starch accumulation in leaves and root tips and 1 000-seed weight of seeds, but decreased soluble sugar content. The results obtained in this study showed that IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ might be important genes regulating sucrose and sugar content traits in sweet potato. They might carry out physiological functions on cell membrane, such as transmembrane transport of sucrose, sucrose into and out of sink tissue, as well as transport and unloading of sucrose into phloem. The changes in traits result from their heterologous expression in Arabidopsis indicates their potential in improving the yield of other plants or crops. The results obtained in this study provide important information for revealing the functions of IbSUT₆₂₇₈₈ and IbSUT₈₁₆₁₆ in starch and glucose metabolism and formation mechanism of important quality traits in sweet potato.

Abstract:Hevea brasiliensis is the main source of natural rubber. Restricted by its tropical climate conditions, the planting area in China is limited, resulted in a low self-sufficiency. Periploca sepium which can produce natural rubber is a potential substitute plant. cis-prenyltransferase (CPT), small rubber particle protein (SRPP) and rubber elongation factor (REF) are key enzymes involved in the biosynthesis of cis-1,4-polyisoprene, the main component of natural rubber. In this study, we cloned the promoter sequences of CPT, SRPP and REF through chromosome walking strategy. The spatial expression patterns of the three promoters were analyzed using GUS (β-glucuronidase) as a reporter gene driven by the promoters through Agrobacterium-mediated genetic transformation. The results showed that GUS driven by CPT, SRPP or REF promoter was expressed in leaves and stems, especially in the leaf vein and vascular bundle. The GUS activity in stems was higher than that in leaf. This study provided a basis for analyzing the biosynthesis mechanism of natural rubber and breeding new varieties of high yield natural rubber.

Abstract:Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.

Abstract:Phenylalanine ammonia-lyase (PAL) is the key entry enzyme of plant phenylpropanoid pathway. It plays an important role in the biosynthesis of podophyllotoxin, an anti-tumor lignan that is currently produced from its main natural source Sinopodophyllum hexandrum (Royle) Ying. In this study, we cloned the gene ShPAL encoding phenylalanine ammonia-lyase by RT-PCR from the root of S. hexandrum ecotype inhabited in the Aba' district, Sichuan, based on its public SRA transcriptome data-package. Bioinformatics analyses showed that the ShPAL-encoded protein is composed of 711 amino acids, contains the conserved domains of PAL, and has the signature motif within the active center of aromatic ammonia-lyases. Moreover, ShPAL protein was predicted to have a secondary structure mainly composed of α-helix and random coil, a typical ‘seahorse’ shape monomer tertiary structure, and a homologous tetramer three-dimensional structure by Swiss-Modelling. The phylogenetic lineage analysis indicated ShPAL was of the highest sequence identity and the shortest evolutionary distance with the PAL of Epimedium sagittatum from the same Berberidaceae family. Subcellular localization experiments showed that ShPAL protein was mainly distributed in the cytoplasm, despite of a minority on the endoplasmic reticulum membrane. Furthermore, ShPAL protein was recombinantly expressed in Escherichia coli and purified by histidine-tag affinity chromatography. Its enzymatic activity was determined up to 20.91 U/mg, with the optimum temperature of 41℃ and pH of 9.0. In contrast, the enzyme activity of its F130H mutant decreased by about 23.6%, yet with the same trends of change with temperature and pH, confirming that phenylalanine at this position does affect the substrate specificity of PAL. Both the wild type and the mutant have relatively poor thermostability, but good pH-stability. These results may help to further investigate the regulatory role of PAL in the process of podophyllotoxin biosynthesis and advance the heterologous synthesis of podophyllotoxin to protect the germplasm resource of S. hexandrum. They also demonstrate that ShPAL has a potential application in biochemical industry and biomedicine.

Abstract:The present study aims to explore the genetic diversity of germplasm resources of Chrysanthemum×morifolium (hereinafter, C.×morifolium) at the molecular level and to establish a fingerprint database of C.×morifolium varieties. We employed 12 pairs of primers with high levels of polymorphism, clear bands, and high degrees of reproducibility to analyze the SSR molecular markers and genetic diversity of 91 C.×morifolium materials and 14 chrysanthemum- related materials. With regard to constructing the fingerprints of the tested materials, we chose 9 pairs of core primers. The findings revealed that 12 primer pairs detected 104 alleles in 105 samples, ranging from 2 to 26. The average number of observed alleles (N_a) per site was 9.25. The average number of effective alleles (N_e) per site was 2.745 6, with its range being 1.276 0 to 4.742 5. Shannon genetic diversity index (I) values ranged between 0.513 3 and 2.239 9 (M=1.209 0). Nei's gene diversity index (H) ranged between 0.216 3 and 0.789 1 (M=0.578 0). The observed heterozygosity (H_o) ranged between 0.223 3 and 0.895 2 (M=0.557 5). The expected heterozygosity (H_e) ranged between 0.217 4 and 0.793 3 (M=0.580 8). The polymorphism information content (PIC) ranged between 0.211 5 and 0.774 0 (M=0.532 9). The genetic similarity (GS) ranged between 0.228 5 and 1.000 0 (M=0.608 3). Cluster analysis revealed that when the genetic distance (GD) equals to 0.30, the tested materials can be classified into 2 groups. When the GD equals to 0.27, the first group can be divided into 6 subgroups; accordingly, 105 tested materials can be divided into 7 subgroups. The cophenetic correlation test was carried out based on the cluster analysis, and the corresponding results showed that the cluster map correlated with the genetic similarity coefficient (r=0.952 73). According to the results of Structure population analysis, we obtained the optimal population number, with the true number of populations (K) being 3 and the population being divided concerning Q ≥ 0.5. Three subgroups, i.e., Q1, Q2 and Q3, included 34, 33 and 28 germplasms, respectively, and the remaining 10 germplasms were identified as the mixed population. During the experiment, 9 pairs of core primers were screened among the total of 12 for a complete differentiation regarding 105 tested materials, and the fingerprints of 91 C.×morifolium materials and 14 chrysanthemum-related materials were further constructed. Overall, there were significant genetic differences and rich genetic diversity among C.×morifolium materials, which would shed light on the garden application and variety selection fields of C.×morifolium. The fingerprint database of 105 C.×morifolium varieties and chrysanthemum-related species may provide technical support for future research regarding the identification and screening system of C.×morifolium varieties.

Abstract:Auto-inhibited Ca²⁺-ATPase (ACA) is one of the Ca²⁺-ATPase subfamilies that plays an important role in maintaining Ca²⁺ concentration balance in plant cells. To explore the function and gene expression pattern of the RcACA gene family in castor, bioinformatics analysis was used to identify the members of the RcACA gene family in castor. The basic physical and chemical properties, subcellular location, protein secondary and tertiary structure, conserved domain, conserved motif, gene structure, chromosome location and collinear relationship, as well as the evolutionary characteristics and promoter cis-acting elements were predicted and analyzed. The expression pattern of the RcACA gene under abiotic stress was analyzed by expression (fragments per kilobase of exon model per million mapped fragments, FPKM) in castor transcriptome data. The results showed that 8 RcACA gene family members were identified in castor, acidic proteins located in the plasma membrane. In the secondary structure of all proteins, the α-helix and random coil is more; the RcACA genes were clustered into three categories, and the design of the genes in the same category was similar to the conserved motif. Both of them had four typical domains, RcACA3-RcACA8 had a Ca²⁺-ATPase N-terminal autoinhibitory domain. The RcACA gene is mostly located on the long arm of the chromosome and has 2 pairs of collinear relationships. There are more light response elements but fewer hormone-induced elements located upstream of the RcACA coding region. Interspecific clustering showed that the evolution of ACA genes among species was conservative. Tissue expression pattern analysis showed that RcACA genes showed apparent tissue expression specificity, and most of the genes showed the highest expression level in male flowers. Expression analysis under abiotic stress showed that RcACA2-RcACA8 were up-regulated under high salt and drought stress, and RcACA1 was up-regulated at 0-24 h under low-temperature stress, indicating that RcACA genes positively responded to abiotic stresses. The above results provide a theoretical basis for exploring the role of the RcACA gene in castor growth, development and stress response.

Abstract:Glutamate receptor-like (GLR) is an important class of Ca²⁺ channel proteins, playing important roles in plant growth and development as well as in response to biotic and abiotic stresses. In this paper, we performed genome-wide identification of banana GLR gene family based on banana genomic data. Moreover, we analyzed the basic physicochemical properties, gene structure, conserved motifs, promoter cis-acting elements, evolutionary relationships, and used real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) to verify the expression patterns of some GLR family members under low temperature of 4℃ and different hormone treatments. The results showed that there were 19 MaGLR family members in Musa acuminata, 16 MbGLR family members in Musa balbisiana and 14 MiGLR family members in Musa itinerans. Most of the members were stable proteins and had signal peptides, all of them had 3-6 transmembrane structures. Prediction of subcellular localization indicated that all of them were localized on the plasma membrane and irregularly distributed on the chromosome. Phylogenetic analysis revealed that banana GLRs could be divided into 3 subclades. The results of promoter cis-acting elements and transcription factor binding site prediction showed that there were multiple hormone- and stress-related response elements and 18 TFBS in banana GLR. RT-qPCR analysis showed that MaGLR1.1 and MaGLR3.5 responded positively to low temperature stress and were significantly expressed in abscisic acid/methyl jasmonate treatments. In conclusion, the results of this study suggest that GLR, a highly conserved family of ion channels, may play an important role in the growth and development process and stress resistance of banana.

Abstract:MADS-box gene family is a significant transcription factor family that plays a crucial role in regulating plant growth, development, signal transduction, and other processes. In order to study the characteristics of MADS-box gene family in Docynia delavayi (Franch.) Schneid. and its expression during different stages of seed germination, this study used seedlings at different stages of germination as materials and screened MADS-box transcription factors from the transcriptome database of D. delavayi using bioinformatics methods based on transcriptome sequencing. The physical and chemical properties, protein conservative motifs, phylogenetic evolution, and expression patterns of the MADS-box transcription factors were analyzed. Quantitative real-time PCR (qRT-PCR) was used to verify the expression of MADS-box gene family members during different stages of seed germination in D. delavayi. The results showed that 81 genes of MADS-box gene family were identified from the transcriptome data of D. delavayi, with the molecular weight distribution ranged of 6 211.34-173 512.77 Da and the theoretical isoelectric point ranged from 5.21 to 10.97. Phylogenetic analysis showed that the 81 genes could be divided into 15 subgroups, among which DdMADS27, DdMADS42, DdMADS45, DdMADS46, DdMADS53, DdMADS61, DdMADS76, DdMADS77 and DdMADS79 might be involved in the regulation of ovule development in D. delavayi. The combination of the transcriptome data and the qRT-PCR analysis results of D. delavayi seeds indicated that DdMADS25 and DdMADS42 might be involved in the regulation of seed development, and that DdMADS37 and DdMADS38 might have negative regulation effects on seed dormancy. Previous studies have reported that the MIKC^* subgroup is mainly involved in regulating flower organ development. For the first time, we found that the transcription factors of the MIKC^* subgroup exhibited a high expression level at the early stage of seed germination, so we speculated that the MIKC^* subgroup played a regulatory role in the process of seed germination. To verify the accuracy of this speculation, we selected DdMADS60 and DdMADS75 from the MIKC^* subgroup for qRT-PCR experiments, and the experimental results were consistent with the expression trend of transcriptome sequencing. This study provides a reference for further research on the biological function of D.delavayi MADS-box gene family from the perspective of molecular evolution.

Abstract:Pellionia scabra belongs to the genus Pellionia in the family of Urticaceae, and is a high-quality wild vegetables with high nutritional value. In this study, high-throughput techniques were used to sequence, assemble and annotate the chloroplast genome. We also analyzed its structure, and construct the phylogenetic trees from the P. scabra to further study the chloroplast genome characteristics. The results showed that the chloroplast genome size was 153 220 bp, and the GC content was 36.4%, which belonged to the typical tetrad structure in P. scabra. The chloroplast genome encodes 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes in P. scabra. Among them, 15 genes contained 1 intron, 2 genes contained 2 introns, and rps12 had trans-splicing, respectively. In P. scabra, chloroplast genomes could be divided into four categories, including 43 photosynthesis, 64 self-replication, other 7 coding proteins, and 4 unknown functions. A total of 51 073 codons were detected in the chloroplast genome, among which the codon encoding leucine (Leu) accounted for the largest proportion, and the codon preferred to use A and U bases. There were 72 simple sequence repeats (SSRs) in the chloroplast genome of P. scabra, containing 58 single nucleotides, 12 dinucleotides, 1 trinucleotide, and 1 tetranucleotide. The ycf1 gene expansion was present at the IRb/SSC boundary. The phylogenetic trees showed that P. scabra (OL800583) was most closely related to Elatostema stewardii (MZ292972), Elatostema dissectum (MK227819) and Elatostema laevissimum var. laevissimum (MN189961). Taken together, our results provide worthwhile information for understanding the identification, genetic evolution, and genomics research of P. scabra species.

Abstract:Dracaena marginata is a widely cultivated horticultural plant in the world, which has high ornamental and medicinal value. In this study, the whole genome of leaves from D. marginata was sequenced by Illumina HiSeq 4000 platform. The chloroplast genome were assembled for functional annotation, sequence characteristics and phylogenetic analysis. The results showed that the chloroplast genome of D. marginata composed of four regions with a size of 154 926 bp, which was the smallest chloroplast genome reported for Dracaena species to date. A total of 132 genes were identified, including 86 coding genes, 38 tRNA genes and 8 rRNA genes. Codon bias analysis found that the codon usage bias was weak and there was a bias for using A/U base endings. 46 simple sequence repeat and 54 repeats loci were detected in the chloroplast genome, with the maximum detection rate in the large single copy region and inverted repeat region, respectively. The inverted repeats boundaries of D. marginata and Dracaena were highly conserved, whereas gene location differences occurred. Phylogenetic analysis revealed that D. serrulata and D. cinnabari form a monophyletic clade, which was the closest relationship and conformed to the morphological classification characteristics. The analysis of the chloroplast genome of D. marginata provides important data basis for species identification, genetic diversity and chloroplast genome engineering of Dracaena.

Abstract:The genomic DNA of Rubus rosaefolius was extracted and sequenced by Illumina NovaSeq platform to obtain the complete chloroplast genome sequence, and the sequence characteristics and phylogenetic analysis of chloroplast genes were carried out. The results showed that the complete chloroplast genome of the R. rosaefolius was 155 650 bp in length and had a typical tetrad structure, including two reverse repeats (25 748 bp each), a large copy region (85 443 bp) and a small copy region (18 711 bp). A total of 131 genes were identified in the whole genome of R. rosaefolius chloroplast, including 86 protein coding genes, 37 tRNA genes and 8 rRNA genes. The GC content of the whole genome was 36.9%. The genome of R. rosaefolius chloroplast contains 47 scattered repeats and 72 simple sequence repeating (SSR) loci. The codon preference is leucine codon, and the codon at the end of A/U is preferred. Phylogenetic analysis showed that R. rosaefolius had the closest relationship with R. taiwanicola, followed by R. rubraangustifolius and R. glandulosopunctatus. The chloroplast genome characteristics and phylogenetic analysis of R. rosaefolius provide a theoretical basis for its genetic diversity research and chloroplast development and utilization.

Abstract:Incarvillea younghusbandii Sprague is a traditional tonic herb. The roots are used as herbal medicine for nourishing and strengthening, as well as treating postpartum milk deficiency and weakness. In this study, the chloroplast genome of I. younghusbandii was sequenced and assembled by the high-throughput sequencing technology. The sequence characteristics, sequence repeats, codon usage bias, phylogenetic relationships and estimated divergence time of I. younghusbandii were analyzed. The 159 323 bp sequence contained a large single copy (80 197 bp), a small single copy (9 030 bp) and two inverted repeat sequences (35 048 bp). It contained 120 genes, including 77 protein coding genes, 8 ribosomal RNA genes and 35 transfer RNA genes. AAA was the most frequent codon in the chloroplast coding sequence of I. younghusbandii. A total of 42 simple sequence repeats were identified in the chloroplast genome. Phylogenetic analysis revealed I. younghusbandii was mostly like its taxonomically close relative Incarvillea compacta. The divergence between I. younghusbandii and I. compacta was dated to 4.66 million years ago. This study was significant for the scientific conservation and development of resources related to I. compacta. It also provides a basic genetic resource for the subsequent species identification of the genus Incarvillea, and the population genetic diversity study of Bignoniaceae.

Abstract:Schizothorax argentatus that only distributes in the Ili River basin in Xinjiang is one of the rare and endangered species of schizothorax in China, thus has high scientific and economic values. In this study, the complete mitochondrial genome sequence of S. argenteus with a length of 16 580 bp was obtained by high-throughput sequencing. The gene compositions and arrangement were similar to those of typical vertebrates. It contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a non-coding region (D-loop). The nucleotide compositions were A (30.25%), G (17.28%), C (27.20%), and T (25.27%), respectively, showing obvious AT bias and anti-G bias. Among the tRNA genes, only tRNA-Ser^(GCU) could not form a typical cloverleaf structure due to the lack of dihydrouracil arm. The AT-skew and GC-skew values of the ND6 gene were fluctuating the most, suggesting that the gene may experience different selection and mutation pressures from other genes. The mitochondrial control region of S. argenteus contained three different domains, i.e., termination sequence region (ETAS), central conserved region (CSB-F, CSB-E, CSB-D, and CSB-B), and conserved sequence region (CSB1, CSB2, and CSB3). The conserved sequence fragment TT (AT) nGTG, which was ubiquitous in Cypriniformes, was identified at about 50 bp downstream CSB3. Phylogenetic relationships based on the complete mitochondrial genome sequence of 28 Schizothorax species showed that S. argenteus had differentiated earlier and had a distant relationship with other species, which may be closely related to the geographical location and the hydrological environment where it lives.

Abstract:The generation of a tau-V337M point mutation mouse model using gene editing technology can provide an animal model with fast disease progression and more severe symptoms, which facilitate the study of pathogenesis and treatment of Alzheimer's disease (AD). In this study, single guide RNAs (sgRNA) and single-stranded oligonucleotides (ssODN) were designed and synthesized in vitro. The mixture of sgRNA, Cas9 protein and ssODN was microinjected into the zygotes of C57BL/6J mice. After DNA cutting and recombination, the site homologous to human 337 valine (GTG) in exon 11 was mutated into methionine (ATG). In order to improve the efficiency of recombination, a Rad51 protein was added. The female mice mated with the nonvasectomy male mice were used as the surrogates. Subsequently, the 2-cell stage gene edited embryos were transferred into the unilateral oviduct, and the F0 tau-V337M mutation mice were obtained. Higher mutation efficiency could be obtained by adding Rad51 protein. The F0 tau-V337M point mutation mice can pass the mutation on to the F1 generation mice. In conclusion, this study successfully established the first tau-V337M mutation mouse by using Cas9, ssODN and Rad51. These results provide a new method for developing AD mice model which can be used in further research on the pathogenesis and treatment of AD.

Abstract:To explore the differentially expressed genes (DEGs) related to biosynthesis of active ingredients in wolfberry fruits of different varieties of Lycium barbarum L. and reveal the molecular mechanism of the differences of active ingredients, we utilized Illumina NovaSeq 6000 high-throughput sequencing technology to conduct transcriptome sequencing on the fruits of ‘Ningqi No.1’ and ‘Ningqi No.7’ during the green fruit stage, color turning stage and maturity stage. Subsequently, we compared the profiles of related gene expression in the fruits of the two varieties at different development stages. The results showed that a total of 811 818 178 clean reads were obtained, resulting in 121.76 Gb of valid data. There were 2 827, 2 552 and 2 311 DEGs obtained during the green fruit stage, color turning stage and maturity stage of ‘Ningqi No. 1’ and ‘Ningqi No. 7’, respectively, among which 2 153, 2 050 and 1 825 genes were annotated in six databases, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) and clusters of orthologous groups of proteins (KOG). In GO database, 1 307, 865 and 624 DEGs of green fruit stage, color turning stage and maturity stage were found to be enriched in biological processes, cell components and molecular functions, respectively. In the KEGG database, the DEGs at three developmental stages were mainly concentrated in metabolic pathways, biosynthesis of secondary metabolites and plant-pathogen interaction. In KOG database, 1 775, 1 751 and 1 541 DEGs were annotated at three developmental stages, respectively. Searching the annotated genes against the PubMed database revealed 18, 26 and 24 DEGs related to the synthesis of active ingredients were mined at the green fruit stage, color turning stage and maturity stage, respectively. These genes are involved in carotenoid, flavonoid, terpenoid, alkaloid, vitamin metabolic pathways, etc. Seven DEGs were verified by RT-qPCR, which showed consistent results with transcriptome sequencing. This study provides preliminary evidences for the differences in the content of active ingredients in different Lycium barbarum L. varieties from the transcriptional level. These evidences may facilitate further exploring the key genes for active ingredients biosynthesis in Lycium barbarum L. and analyzing their expression regulation mechanism.

Abstract:Protein Engineering is a core compulsory course of biotechnology major, which is the first-class undergraduate major being constructed in Shanxi Province. In view of the problems of single teaching mode of Protein Engineering, such as insufficient students' participation, short teaching time, and expensive experiment cost, the course team carried out the reform and practice of teaching mode for this course, and put forward a new teaching strategy. Under the guidance of the "Golden Course" standard for advancement, innovation and challenge, the course team developed the materials for massive open online courses (MOOC), and carried out the online and offline mixed teaching of Protein Engineering based on BOPPPS+flipped classroom by using the Chao-Xing Fan-Ya network teaching platform. Through this, a comprehensive, systematic and dynamic new teaching system of Protein Engineering was developed. Using the teaching mode based on BOPPPS+flipped classroom, the offline classroom teaching was combined with students' online self-study and homework completion, chapter test and discussion, and this mixed teaching mode was fully integrated into the flipped classroom. After three rounds of teaching practice, the course team had developed a complete, reproducible, scientific and reasonable online and offline mixed teaching mode, which included course materials preparation, exploring experiment guidance, classroom discussion design and course performance evaluation. The online and offline mixed teaching mode of Protein Engineering based on BOPPPS+flipped classroom was helpful for students to improve their autonomous learning ability, to be deeply engaged in the whole teaching process, and to develop a comprehensive and profound understanding of Protein Engineering. This teaching mode improved the teaching quality of Protein Engineering, and facilitated students to learn other follow-up professional courses. Moreover, it provides a reference for the course teaching reform.